research article prolonged hypocalcemic effect by...

Research ArticleProlonged Hypocalcemic Effect by PulmonaryDelivery of Calcitonin Loaded Poly(Methyl Vinyl EtherMaleic Acid) Bioadhesive Nanoparticles

J. Varshosaz,1 M. Minaiyan,2 and M. Forghanian1

1 Department of Pharmaceutics, School of Pharmacy and Novel Drug Delivery Systems Research Centre,Isfahan University of Medical Sciences, P.O. Box 81745-359, Isfahan 81746-73461, Iran

2Department of Pharmacology, School of Pharmacy, Isfahan University of Medical Sciences, Isfahan 81746-73461, Iran

Correspondence should be addressed to J. Varshosaz; [email protected]

Received 9 September 2013; Revised 8 December 2013; Accepted 12 January 2014; Published 20 February 2014

Academic Editor: Fabio Sonvico

Copyright © 2014 J. Varshosaz et al. This is an open access article distributed under the Creative Commons Attribution License,which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

The purpose of the present study was to design a pulmonary controlled release system of salmon calcitonin (sCT). Therefore,poly(methyl vinyl ether maleic acid) [P(MVEMA)] nanoparticles were prepared by ionic cross-linking method using Fe2+ andZn2+ ions. Physicochemical properties of nanoparticles were studied in vitro. The stability of sCT in the optimized nanoparticleswas studied by electrophoretic gel method. Plasma calcium levels until 48 h were determined in rats as pulmonary-free sCTsolution or nanoparticles (25𝜇g⋅kg−1), iv solution of sCT (5 𝜇g⋅kg−1), and pulmonary blank nanoparticles.The drug remained stableduring fabrication and tests on nanoparticles.The optimized nanoparticles showed proper physicochemical properties. Normalizedreduction of plasma calcium levels was at least 2.76 times higher in pulmonary sCT nanoparticles compared to free solution. Theduration of hypocalcemic effect of pulmonary sCT nanoparticles was 24 h, while it was just 1 h for the iv solution. There wasnot any significant difference between normalized blood calcium levels reduction in pulmonary drug solution and iv injection.Pharmacological activity of nanoparticles after pulmonary delivery was 65% of the iv route. Pulmonary delivery of P(MVEMA)nanoparticles of sCT enhanced and prolonged the hypocalcemic effect of the drug significantly.

1. IntroductionCalcitonin (also known as thyrocalcitonin) is a polypeptidehormone of 32 amino acids, with a molecular weight of3454.93 Daltons (approximately 3.5 kDa). Its structure iscomprised of a single alpha helix. CT hormone is producedin humans primarily by the parafollicular cells of the thyroid,and in many other animals in the ultimobranchial body. Itacts to reduce blood calcium (Ca2+), through opposing theeffects of parathyroid hormone (PTH) [1–3]. Calcium playsan important role in most of cell activities. It contributes inmuscles contraction and materials metabolism intracellular,and in bones, blood coagulation, and the transition of ner-vous signals extracellular [4, 5]. CT is found widely through-out different species such as fish (salmon), reptiles, birds, andmammals, while there are several differences in amino acidcomposition of CT from different sources which is associatedwith different potencies. Salmon calcitonin (sCT) is widely

used because it is the most potent CT available with goodtolerability. Currently, it is produced either by recombinantDNA technology or by chemical peptide synthesis [6–8].This hormone acts both directly on osteoclasts, resulting ininhibition of bone resorption, and directly on chondrocytes,attenuating cartilage degradation and stimulating cartilageformation [9]. CT lowers blood Ca2+ levels in three ways: (1)inhibiting Ca2+ absorption by the intestines, (2) inhibitingosteoclasts activity and activating osteoblasts in bones, (3)inhibiting renal tubular cell reabsorption of Ca2+ allowing itto be secreted in the urine. However, the effect of CT thatmirrors those of PTH is inhibition of phosphate reabsorptionby the kidney tubules [10–14]. It seems that CT hormone hasimportant effects on bone amendment, especially spinal col-umn, then sCT is used for the treatment of postmenopausalosteoporosis, hypercalcaemia, Paget’s disease, bone metas-tases, and phantom limb pain [15].

Hindawi Publishing CorporationBioMed Research InternationalVolume 2014, Article ID 932615, 13 pageshttp://dx.doi.org/10.1155/2014/932615

2 BioMed Research International

Bioavailability of sCT following subcutaneous and intra-muscular injection in humans is high and similar for the tworoutes of administration (71% and 66%, resp.). Plasma proteinbinding is 30% to 40%, following parenteral administrationof 100 IU sCT and its peak plasma concentration lies betweenabout 200 and 400 pg mL−1 [16].

Currently, sCT from salmon species is commercially avai-lable in the formof injection and intranasal spray (Miacalcin).Data on the bioavailability of Miacalcin that is rapidlyabsorbed from nasal mucosa shows great variability with anaverage bioavailability of 3% compared to injection form [17,18].Themaximal sCTplasma concentration is reachedwithin31–39min following nasal administration of sCT solution.Moreover, sCT elimination half-lives of this way are 43minutes [19].

Due to its short half-life (approximately 15–20min) afterparenteral administration, frequent administrations are req-uired to maintain its pharmacological effects [19]. Moreover,in effective inhibition of the clinical symptoms of metabolicbone disorders such as osteoporosis or Paget’s disease, arelatively high dosage of sCT is administered. Calcitoninmayalso be as an alternative for patients who cannot take the bis-phosphonate drugs. However, the daily injection of sCT haspoor patient compliance for long-term therapy. Therefore,controlled release delivery systems of this drug have attractedthe interest of pharmaceutical researchers. Currently, themost common delivery route to administer sCT is throughintramuscular or intravenous injections. Various researchershave studied different routes to deliver sCT more effectivelyand to decrease frequent administrations, including oral [20,21], nasal [22, 23], vaginal [24], and rectal [25, 26] delivery.These systems are designed to protect the polypeptide againstthe biological barriers.

Petersen et al. [27] attempted to enable intestinal sCTabsorption in rats using themild surfactant, tetradecylmalto-side (TDM) as an intestinal permeation enhancer. TDMcaused an increased absolute bioavailability of sCT in ratcolon from 1.0% to 4.6%, whereas no enhancement increasewas observed in ileal and jejunal instillations.

In another study, sCT-containing microspheres weredesigned for colonic delivery by applying a pH-sensitivepolymer Eudragit P-4135F which showed a four-fold increaseof the area above the curve of calcium blood levels comparedto levels reached by sCT solution after 8–12 h [20].

Another polymer that was used in the production ofsCT microcapsules was biodegradable poly(DL-lactic acid)[28]. Poly(lactide-co-glicolide) (PLGA) microspheres [29,30], polyisobutylcyanoacrylate nanoparticles [31], and poly(ethylene glycol)-terephthalate (PEGT)/poly(butylene tere-phthalate) (PBT) [32] are also among the other reported bio-degradable polymers used for controlled and sustained deliv-ery of sCT.Absorption enhancers such as hydroxypropyl- anddimethyl-beta-cyclodextrins [33], the bile salts, chitosan, andloryl carnitinwere evaluated for enhancing absorption of sCTfrom nasal route in rats [34].

Moreover, nanoparticles of glycol chitosan and its thio-lated derivative resulted in a pronounced hypocalcemic effectfor at least 12 and 24 h and a corresponding pharmacolog-ical availability of 27 and 40%, respectively, by improving

the pulmonary mucoadhesive properties [35]. Relative lungbioavailability of these nanoparticles ranged from 11% to 18%.All formulations were prepared by spray drying powderscontaining sCT, human serum albumin, mannitol, and citricacid/sodium citrate [36].

Pulmonary administration of elcatonin loaded in chi-tosan-modified PLGA nanospheres resulted in more slowlyelimination of drug from the lungs and reduced bloodcalcium levels to 80%with prolonged pharmacological actionfor 24 h which was significantly longer than that by chitosan-unmodified PLGA nanospheres. The absorption-enhancingeffect may have been caused by opening the intercellular tightjunctions [37].

The sCT powder suitable for inhalation was preparedusing a spray-drying process which contained chitosan andmannitol as absorption enhancer and protecting agents.

Although some studies have been on the pulmonarydelivery of sCT, there is not any pulmonary marketed dosageform of this drug yet. Consequently, further studies on moreversatile drug delivery systems of this drug are necessary toincrease its bioavailability. In the present study, bioadhesivenanoparticles of sCT were developed for pulmonary deliveryusing P(MVEMA) as a mucoadhesive polymer [39, 40] toincrease the absorption of the drug in lungs mucosa.

Poly(methyl vinyl ether-alt-maleic acid) is awater-solublepoly anion having molecular masses ranging from 20 to1980 kDa (average 522.45 kDa).The nanoparticles made fromanhydride form of this copolymer have shown strong bioad-hesive interactions with components of the gut mucosa [41–43].

At themoment there is not any pulmonary dosage formofsCT in the market and the present intranasal and intramus-cular forms have only 3 and 5% bioavailability, respectively,with half-lives of less than 1 hour. The low bioavailability andshort half-life of this drug have caused frequent need for drugadministration which increases the cost of the therapy andreduces the patient compliance. For these reasons, the aimof the present study was to produce inhalable nanoparticlesof sCT so as to increase the duration of hypocalcemic effectof this drug and enhance its bioavailability by its deliverythrough the pulmonary route of administration.

2. Materials and Methods

2.1. Chemicals and Reagents. sCT (Limhamn, Sweden) wasused as the active ingredient. Poly(methyl vinyl ether-alt-maleic acid), mucin, and Coomassie Brilliant Blue G-250dye were purchased from Sigma Chemical Company (USA).Zinc sulfate, iron chloride, ethanol, methanol, hydrochloricacid, and NaOH were purchased from Merck ChemicalCompany (Germany). Orthophosphoric acid 85%w/v wasfrom Panreac (Spain).

2.2. Preparation of sCT Loaded P(MVEMA) Nanoparticles.A two-level factorial design was used for preparation ofsCT loaded nanoparticles.Three different variables includingstirrer rate (400 or 1000 rpm), cross-linking ion type (Fe+2or Zn+2), and curing time (5 or 20min) were studied and 8different formulations (Table 1) were designed.

BioMed Research International 3

Table 1: Studied variables in factorial design used in preparation ofsCT loaded in P(MVEMA) nanoparticles.

Studied variables LevelsII I

Stirring rate (rpm) 400 1000Ion type Zn2+ Fe2+

Curing time (min) 5 20

Table 2: Composition of different formulations of sCT loadedP(MVEMA) nanoparticles designed by a two levels factorial design.

Formulationcode

Stirring rate (𝑅)(rpm) Ion type (𝐼) Curing time (𝐶)

(min)𝑅1000𝐼Fe𝐶5 1000 Fe 5.00

𝑅400𝐼Fe𝐶5 400 Fe 5.00

𝑅400𝐼Zn𝐶20 400 Zn 20.00

𝑅1000𝐼Zn𝐶5 1000 Zn 5.00

𝑅1000𝐼Zn𝐶20 1000 Zn 20.00

𝑅400𝐼Zn𝐶5 400 Zn 5.00

𝑅1000𝐼Fe𝐶20 1000 Fe 20.00

𝑅400𝐼Fe𝐶20 400 Fe 20.00

Nanoparticles were prepared by an ionic cross-linkingmethod of P(MVEMA) [42] to obtain optimized nanoparti-cles with suitable particle size, zeta potential, drug loadingefficiency, drug release efficiency, and mucoadhesive prop-erties. In a typical procedure, 250mg of P(MVEMA) wasdissolved in 5mL of deionized water at room temperatureon a magnetic stirrer (IKA-WERKE, model RT 10 Power,Japan) with the speed of 450 rpm, and after 20 minuteswhen it dissolved completely, the pH of the solution wasadjusted on 7.4 by adding NaOH. Afterwards, 2.5mg ofsCT was added to this solution while being stirred. In thenext step, a 5% solution of FeCl

2or ZnSo

4(125mg of

the salt in 2.5mL of deionized water) was prepared. Thesolution of the salts was added dropwise to the polymer/drugsolution by an insulin syringe in various stirring rates(according to Table 1) while being stirred at different speeds.An overview of the investigated formulations is presented inTable 2.

A run involved the corresponding combination of lev-els to which the factors in the experiment were set. Allexperiments were carried out in triplicate. The effects of thestudied variables on the responses were then analyzed bythe Design Expert statistical software (version 7.0.0, Stat-Ease, Inc., Minneapolis, MN, USA) to obtain independentlythe main effects of these factors, followed by the analysisof variance (ANOVA) to determine which factors werestatistically significant.

2.3. Preparation of Bradford Reagent. The Bradford proteinassay is a colorimetric or spectroscopic analysis procedurefor determining the concentration of protein in solution. It

involves the binding of dye Coomassie Brilliant Blue G-250dye to proteins in acidic solution. The maximum absorbancefor this solution of Coomassie Brilliant Blue G-250 dye shiftsfrom 465 nm (red or cationic unbound form) to 595 nm (blueor anionic bound form) when binding to protein occurs.For assay of protein concentration, maximum absorbanceof sample solutions was measured in 𝜆max = 595 nm andprotein concentration of sampleswas determined by standardcurve of special protein. The Bradford assay is linear overa short range, typically from 0 to 2000𝜇gmL−1 [43, 44]. Inthe present paper on the preparation of Bradford solution(5x), briefly, 125mg of dark purple powder of CoomassieBrilliant Blue G-250 was dissolved in 62.5mL of 96% ethanolormethanol on stirrer, and 125mL orthophosphoric acid 85%w/v was added to this solution. The resulting solution wasdiluted with water to a final volume of 250mL while the pHwas less than 4.

For measuring the protein concentration of the samplestaken from the medium of release test and also in mea-suring drug loading efficiency in the nanoparticles, 200 𝜇Lof Bradford reagent (5x) was vortexed with 800 𝜇L of eachsample, after about 5 minutes and samples absorbance wasmeasured spectrophotometrically (RF-5301 PC, Shimadzu,Kyoto, Japan) at room temperature in 𝜆max = 595 nm [45].Blank samples were prepared by adding 800𝜇L of deionizedwater to 200𝜇L of Bradford reagent.

2.4. Standard Curve of sCT. Solutions with different concen-trations of 2.5, 7.5, 15, 22.5, 30, 45, 60, and 75 𝜇gmL−1 of sCTwere prepared by dilution of a 100 𝜇gmL−1 stock solution ofthis drug in deionized water. Then, the absorbance of eachsolution was measured spectrophotometrically at 𝜆max =595 nm by Bradford method. This procedure was repeatedthree times for each concentration and in three consecutivedays.

2.5. Particle Size and Zeta Potential Determination. Themeanparticle size (z-average) and zeta potential of sCT nanoparti-cles were determined by Zetasizer (Zetasizer 3600, MalvernInstrument Ltd., Worchestershire, UK) at 25∘C. All particlesize measurements were performed in deionized water usinga He-Ne laser beam at 658 nm with a scattering angle of 130∘without dilution.

2.6. Entrapment Efficiency Determination. The entrapmentefficiency percent (EE%) was determined after centrifuga-tion (Eppendorf AG 23331, model 5430, Hamburg, Germany)of nanoparticles dispersion using Eppendorf tubes (Ami-con Ultra, Ireland, and cut-off 10000Da) by measuring theconcentration of free drug in aqueous medium. For thispurpose, 1mL of the drug loaded nanoparticles was cen-trifuged at 10000 rpm for 15min; then, the concentra-tion of free drug in the filtrate was measured after addingBradford reagent at 𝜆max = 595 nm. The concentrationof loading drug was calculated indirectly by calculating thedifference between the initial concentrations of the drugused (2.5mg/7.5mL) and the concentration of free drug in


the aqueous medium by Bradford method. The EE% wascalculated using

Drug loading efficiency or EE%

=Drugtotal − Drugfiltrate

Drugtotal× 100.

(1)

To ensure that the drug has not been absorbed by the filter,a control solution of the drug with similar concentrationof drug in the nanoparticles was prepared and was cen-trifuged in the same time and speed as mentioned earlier.Then the concentration of free drug was determined by thesame method as nanoparticles. The recovery percent of thedrug was 99.4% which showed that almost no importantabsorption of the drug has happened.

In the present study, the indirect method was selected todetermine the amount of drug entrapped in nanoparticle sus-pension as the direct methods for determining nanoparticleEE are always complex and time-consuming. For this purposeafter separating the free drug from the loaded nanoparticlesby centrifugation, the nanoparticles were soaked in aqueousmedium and the dispersion was stirred at 500 rpm for 24hours.Then the amount of released drugwasmeasuredwhichwas in good agreement with the indirect method. Therefore,for saving time in all other experiments the indirect methodwas used for measuring the drug loading efficiency.

2.7. In Vitro Release of sCT from Nanoparticles. Drug releaseprofiles were monitored in phosphate buffer saline (PBS,0.01M, pH 7.0) at 37∘C. 1.5mL of nanoparticles dispersionwas placed in dialysis membrane bags (Mw cutoff 12000Da,Membra-Cel, Viskase, USA). Prior to the release experimenta dialyzing step was carried out by placing dialysis bagin aqueous medium for about 0.5 h to separate free drug.After that, the bags were suspended in a beaker containing7.5mL of PBS under stirring at a speed of 500 rpm. Atappropriate time intervals (0.5, 1, 2, 5, 20, and 24 h) theconcentration of sCT released in themediumwas determinedafter adding Bradford reagent by UV spectrophotometrymethod at 𝜆max = 595 nm. Blank samples were tested by thesame procedure. Release efficiency within 24 h (RE

24%) was

used to compare the release profiles and was calculated using

RE24% =∫𝑡

0𝑦 ⋅ 𝑑𝑡

𝑦100 ⋅ 𝑡× 100. (2)

2.8. Mucoadhesion Measurement of Nanoparticles. Briefly,a stock solution (1mgmL−1) of mucin was prepared withacetate buffer (pH 4.5). One mL of the nanoparticles disper-sion (containing 50mgmL−1) and 2.5mL of mucin solutionwere mixed, vortexed, and shaken at 37∘C for 3 h. The ratioof the nanoparticles to mucin was 20 to 1. Then 1mL ofthis solution was centrifuged and the UV absorbance of thecentrifuged solution was measured after adding Bradfordreagent byUV spectrophotometrymethod at 𝜆max = 595 nm.The concentration of mucin adsorbed to the nanoparticleswas determined by the standard curve of mucin and the

difference between the total amount of the mucin used andthe free mucin after centrifuging was calculated.

To plot the standard curve of mucin, specific concen-trations (100, 200, 300, 400, and 500 𝜇gmL−1) of mucin inacetate buffer (pH 4.5) were prepared by diluting the stocksolution (1000 𝜇gmL−1) and their absorbance was measuredat 𝜆max = 595 nm by adding Bradford reagent. All steps werecarried out using the same method as sCT standard curve.

2.9. Morphology Study. The image of the smallest and thelargest sCL loaded nanoparticles was characterized by scan-ning electron microscope (SEM) (ZEISS Company, contai-ning EBSD system, Germany). The nanoparticles were pri-marily coated with a thin layer of Au/Pd after they weremounted on aluminum stubs and then were examined usingan SEM instrument.

2.10. SDS-PAGE Assay. For evaluation the stability of sCTin optimized formulation during production and release testafter 24 h SDS-page or gel electrophoresis method usingprecast gradient gels (20% Tris-HCl/glycine, Bio-Rad, USA)was used which separates proteins based on their molecularweight range. Appropriate volume (10–20𝜇L) of unstainedmolecular weight protein marker (SMO431, Thermo 26610,Fermenta, Germany) containing proteins with molecularweights such as 116.0, 66.2, 45.0, 35.0, 25.0, 18.4, and 14.4 kDa;a sample taken from the release dialysis solution of the opti-mized nanoparticles after 24 h; a blank sample taken from the24 h release medium of the same nanoparticles formulationbut without drug; and a standard sample of sCT solutionwere pipetted into the SDS-page gel. The mixture of 10 𝜇L ofeach sample and 10 𝜇L of Laemmli sample buffer (62.5mMTris-HCl, pH 6.8, 25% glycerol, 2% SDS, 0.01% Bromophenolblue) with 8% 𝛽-mercaptoethanol was used before runningthe gel and then it was floated 5 minutes in the boiling bathprior to loading. Afterward, the loaded gel was placed inelectrophoretic tank (Bio Rad, USA) and electrophoresedfor 2 h at 100V and then stained with Coomassie blue tovisualize the protein bands. After 2 h it was rinsed and placedin destaining solution for 4 h. This step was repeated severaltimes until area of the gel was completely transparent, and thebands were clearly visible.

2.11. In Vivo Evaluation of sCT Loaded Nanoparticles

2.11.1. Animals. MaleWistar rats, 9 weeks old (weighing 185–330 g) from the animal house of the School of Pharmacyand Pharmaceutical Sciences of IsfahanUniversity ofMedicalSciences, were used to study the pharmacological effectsof pulmonary administered sCT nanoparticles. The animalswere fasted for 12 h (overnight) but had free access to waterimmediately before the experiment. All animals were main-tained under conventional housing conditions (temperature22 ± 2

∘C, relative humidity 55 ± 5%, fresh food, and ina 12:12 h light/dark cycle). Before drug administration, ratswere weighed accurately for calculation of test doses. Theanimal study was approved by the guideline of the ethicalcommittee of Isfahan University of Medical Sciences.


Table 3: Physicochemical properties of P(MVEMA) nanoparticles loaded with sCL.

Formulation code Particle size(nm)

Zeta potential(mV)

Drug loadingefficiency (%)

Drug releaseefficiency after 24 h

(RE24 %)

Mucoadhesiveefficiency (%)

𝑅1000𝐼Fe𝐶5 449.4 ± 3.8 −21.8 ± 1.8 90.0 ± 3.5 44.8 ± 2.7 90.64 ± 0.04

𝑅400𝐼Fe𝐶5 381.4 ± 16.1 −19.7 ± 2.4 88.5 ± 3.0 52.3 ± 4.9 90.66 ± 0.04

𝑅400𝐼Zn𝐶20 227.4 ± 6.6 −19.1 ± 1.0 91.0 ± 1.0 42.3 ± 1.5 90.64 ± 0.04

𝑅1000𝐼Zn𝐶5 206.8 ± 10.8 −19.0 ± 1.4 92.0 ± 3.0 42.6 ± 0.9 90.71 ± 0.05

𝑅1000𝐼Zn𝐶20 189.2 ± 8.4 −18.3 ± 1.1 86.0 ± 2.0 49.2 ± 2.5 90.65 ± 0.06

𝑅400𝐼Zn𝐶5 166.5 ± 1.0 −22.3 ± 0.6 93.0 ± 0.5 46.2 ± 0.5 90.65 ± 0.03

𝑅1000𝐼Fe𝐶20 553.8 ± 20.5 −17.4 ± 0.4 87.0 ± 2.0 51.0 ± 2.3 90.65 ± 0.04

𝑅400𝐼Fe𝐶20 349.6 ± 6.9 −14.6 ± 0.8 88.0 ± 3.5 54.4 ± 2.9 90.66 ± 0.06

2.11.2. Pulmonary and Intravenous Administration. In vivopharmacological studies were performed on 28 rats. The ratswere randomly divided into 4 groups each containing 7 rats.Initially, they were anesthetized by inhalation of ether for abrief period of 10–20 s. The mean sCT dose for pulmonaryadministration was considered to be 25 𝜇g kg−1 [36] and forintravenous (iv) administration it was considered as muchas 5 𝜇g kg−1 to compare the effectiveness of the preparedsCT nanoparticles with the free drug that was used as iv orpulmonary. The sCT solution for iv injection was preparedby dissolving 50 𝜇gmL−1 of sterile distilled water. Then, thesCT solution (group 1) was injected into the lateral tail veinlocated on either side of the tail with 5 𝜇g kg−1 sCT in asingle dose as iv administration. Animals were placed on arechargeable heat pack or circulating warmwater pad to keepthem warm during anesthesia. Under the same conditions,sCT solution was administered pulmonary in the dose of25 𝜇g kg−1 (group 2). Rats of group 3 were administeredblank nanoparticles dispersion as negative control group andthe fourth group received the optimized sCT nanoparticlesdispersion with the dose of 25𝜇g kg−1 according to theloaded sCT. Pulmonary drug administration to the lungsof anesthetized rats was done directly in a single doseusing a 1A 1B microsprayer (Penncentury, USA) device thatgenerates an aerosol by an atomizer-attached syringe. Thisdevice avoids surgical manipulation of animals and allows fora much deep and uniform deposition of the spray droplets inthe lung. Anesthetized animalswere placed in supine positionon a 45∘ slanted support. After the administration, the ratswere maintained in a headup position at an angle of 90∘ tohorizon for 30 s.

2.11.3. Measurement of Plasma Calcium Levels. The hypocal-cemic effect of the sCT on the treated groups of rats wasstudied. Blood samples of approximately 250 𝜇L were col-lected via the retro-orbital sinus vein after anesthetizing ratswith ether, by heparinized capillary at different time pointsuntil 24 h after treatment. At time zero and then at 0.5, 1,3, 12, and 24 h after drug administration, blood samplingwas carried out to determine the calcium concentration. Theplasma was separated by centrifugation at 3000–4000 rpmspeed for 10minutes and total plasma calcium concentrations

were estimated using a commercial photometric availablecalcium kit (Pars Azmon calcium commercial kit for calciumliquid test (CPC), Iran). The assay was carried out with2 𝜇L of plasma after adding 1000 𝜇L of the mixture of tworeagents of the kit. The absorbance of the plasma sampleswas measured at 𝜆max = 570 nm immediately by a UV-VISspectrophotometer (UV mini-SHIMADZU, model 1240CE,Japan) and then changed to concentration using a standardcurve. All assays were run as duplicates with a standard curvefor each assay.

The 48 h area under the reduction percentage of bloodcalcium level-time (h) curve (AUC

0−48) (3) was used for com-

parison of blood calcium lowering effect of sCT developednanoparticles with iv route of administration and pulmonarydelivery of sCT solution:

AUC0−𝑡= ∫

𝑡

0

𝑦𝑑𝑡 (3)

Pharmacological activity (PA) of sCT nanoparticlesadminis-tered from the pulmonary route was calculated from (4):

Pharmacological activity

=dose of 𝑖V solution of sCT × AUCof pulmonary sCT nanoparticles

dose of pulmonary sCT nanoparticles × AUC of 𝑖V solution of sCT

× 100.

(4)

All the in vivo data were analyzed using the one-way analysisof variance (ANOVA) followed by the LSDpost hoc test (SPSS11.5, USA).𝑃 values less than 0.05 were considered significantfor all statistical tests.

3. Results and Discussion

3.1. Particle Size. Theresults of physicochemical properties of8 different formulations of nanoparticles loaded with sCT areshown in Table 3.

Particle size of the nanoparticles changed between 166.5and 553.8 nm (Table 3). The pulmonary delivery of nanopar-ticles may be challenged by a number of physicochemicaland physiological parameters that influence the inhalationdeposition in the lungs. Small particles in the size range of


0

20

40

60

80

100

R-sti

rrin

gra

te

I-io

n ty

pe

C-cu

ring

time RI RC IC

Con

trib

utio

n (%

)

Studied variables

Partial size (nm)Zeta potential (mV)Drug loading efficiency (%)

Drug release efficiency (RE24%)Mucoadhesion efficiency (%)

Figure 1: Contribution of different effective factors on the particlesize, zeta potential, drug loading efficiency, drug release after24 h, and mucoadhesion efficiency of sCT loaded P(MVEMA)nanoparticles.

0.1–1.0 𝜇m are inspired in the alveoli but also are exhaledwithout being deposited significantly [46]. Nebulization ofnanoparticles suspension as aerosol droplets [47] ormicroen-capsulation of them as dry powder inhalers [48] that areappropriate for pulmonary administration are reported toenhance the handling and delivery of nanocarriers.

Analysis of the results of Table 3 by Design Expertsoftware using a two-level factorial design indicated thatthe most effective parameter on the particle size of the sCTnanoparticles was the ion type (Figure 1).

Changing the ion type from Zn to Fe (Figure 2) increasedthe particle size of nanoparticles effectively (𝑃 < 0.05). Whilethese ions have the same charge (+2), the small differencein the effective mean ionic radius of iron (78 pm) and zinc(74 pm) [49] may cause cross-linking of the polymer withFe+2 more effectively than Zn+2. This may be the reasonof greater particle size of nanoparticles prepared by Fe+2compared to Zn+2. Figure 2 shows the other two param-eters had no significant effect on the particle size of thenanoparticles (𝑃 > 0.05). However, increasing the stirringrate increased the particle size of nanoparticles. This may bedue to increased contacts between P(MVEMA) nanoparticlesand strong adhesion properties of P(MVEMA) polymeraggregated the nanoparticles.

3.2. Zeta Potential of Nanoparticles. Zeta potential of thenanoparticles varied between −14.6 and −22.3mV (Table 3).The contribution effect of different studied parameters on thezeta potential of the nanoparticles is shown in Figure 1. Thisfigure shows the most effective factor on zeta potential ofnanoparticles was the curing time (𝑃 < 0.05) and also to alower extent the interaction of the stirring rate and ion type(RI). Figure 2 shows that increasing the curing time increasedthe value of zeta potential (or decreased its absolute value)(𝑃 < 0.05). The prolongation of curing time caused more

covalent bonds and electrical contacts between positivelycharged zinc or iron ionswith P(MVEMA) containingCOO−groups with a negative charge. In fact, with increasing thecross-linking reaction of the polymer more COOH groupsof the polymer are neutralized by the positive chargedions. Therefore, increasing the curing time decreased thenegative charge of the nanoparticles. The decrease in themagnitude of the zeta potential suggested that P(MVEMA)chains partially shielded electric charges of the COOHgroups of the polymer chains in nanoparticles. Sakuma etal. [50, 51] also reported a similar effect for poly(N,N-l-lysinediylterephthaloyl) microcapsules. Changing the levelsof other studied variables, the ion type and stirring rate, didnot have significant effect on the zeta potential (Figure 2)(𝑃 > 0.05).

3.3. Loading Efficiency of sCT in the Nanoparticles. Loadingefficiency of sCT in the nanoparticles changed between 87and 93% (Table 3). Analysis of data in Table 3 revealed thatthe most effective factor on drug loading efficiency was thecuring time (Figure 1). Figure 2 shows almost that changingall the studied parameters (stirring rate, ion type, and curingtime) from level 1 to level 2 decreased the drug loadingin nanoparticles, but it was not statistically different (𝑃 >0.05). The reason for reduction of drug loading efficiency bychanging the ion type from Zn+2 to Fe+2 may be describedby the changes in the particle size of the nanoparticles.Increasing the nanoparticles size by changing the ion type(Figure 2) is believed to be due to the fact that the higheraggregation of polymer together with the connection to thecross-linking agent caused to fill the spaces between thepolymer and the ions or close the open spaces of the polymerchain and thickening of nanoparticles wall. In fact, the overallrigidity of the polymer chain increased and consequentlythe polymer became less permeable. This in turn couldresult in decreasing the drug loading efficiency. Moreover,the higher cross linking ability of Fe2+ may be the cause oflower loading efficiency in nanoparticles prepared by the Fe2+ions. According to the amino acids sequences of the sCT,this drug has a partial positive charge (2 Lys+ amino acids,1 Arg+ amino acid, 1 Glu− amino acid) [52] and therefore thecharge density of Fe2+ or Zn2+may result in a slight repulsionbetween sCT and the ions.Thismay be the reason for loadingefficiency less than 100% in the nanoparticles.

3.4. In Vitro Release of sCT from Nanoparticles. Figure 3shows the release profiles of sCT for different studied for-mulations. This figure shows that release profile of the mostformulations lasted for prolonged time and released approx-imately 80–90% of the loaded drug during 24 h. However,𝑅400𝐼Fe𝐶20 and 𝑅400𝐼Zn𝐶20 had the highest and the lowest

drug release efficiency, respectively (Table 3). Table 4 showsthe correlation coefficients of different release profiles ofall formulations which better fit with a zero-order kineticmodel.

Theprolonged release of sCT fromdifferent nanoparticlesmay be attributed to the biodegradability of the polymer.However, the high concentration of the polymer solution


Stirring rate (rpm) Ion type (Zn or Fe) Curing time (min)

Part

icle

size

(nm

)

450

350

250

150

Zeta

pot

entia

l (m

V)

−15.2

−18.2

−21.2

−24.2

Load

ing

effici

ency

(%)

92

88

84

80

Dru

g re

leas

ed (%

)

55

50

45

40

Muc

oadh

esio

n (%

)

94

92

90

88

100 400 700 1000 Zn Fe 0 5 10 15 20

100 400 700 1000 Zn Fe 0 5 10 15 20

100 400 700 1000 Zn Fe 0 5 10 15 20

100 400 700 1000 Zn Fe 0 5 10 15 20

100 400 700 1000 Zn Fe 0 5 10 15 20

Figure 2: Effect of different levels of studied parameters on the particle size, zeta potential, loading efficiency drug release efficiency, andmucoadhesion ofsCT loaded P(MVEMA) nanoparticles.

and cross-linking agent may be responsible for the sus-tained release property of nanoparticles. This conclusion isconsistent for both ion types (Figure 3). Drug release afterabout 24 h from Zn containing nanoparticles (Figure 3(a))was almost similar to Fe ones (Figure 3(b)).

Figure 1 shows that although drug release was almostmore affected by the ion type but it was not significant (𝑃 >0.05). Changing the two other studied parameters, that is, iontype and curing time, from level 1 to level 2 increased the drugrelease from nanoparticles but not significantly (Figure 2)


0

20

40

60

80

100

120

0 4 8 12 16 20 24 28

Calc

itoni

n re

leas

ed (%

)

Time (h)

R1000IZnC20

R400IZnC5

R400IZnC20

R1000IZnC5

(a)

0

20

40

60

80

100

120

0 4 8 12 16 20 24 28

Calc

itoni

n re

leas

ed (%

)

Time (h)

R400IFeC20

R400IFeC5

R1000IFeC20

R1000IFeC5

(b)

Figure 3: sCT release profiles from P(MVEMA) nanoparticles containing (a) Zn or (b) Fe ion (𝑛 = 3).

Table 4: Correlation coefficients of different kinetic modelsobtained by curve fitting method to sCT release data from differentnanoparticles.

Formulation code First-order Zero-order Higuchi model𝑅1000𝐼Fe𝐶5 0.921 0.986 0.952

𝑅400𝐼Fe𝐶5 0.936 0.986 0.952

𝑅400𝐼Zn𝐶20 0.956 0.987 0.959

𝑅1000𝐼Zn𝐶5 0.935 0.983 0.967

𝑅1000𝐼Zn𝐶20 0.967 0.985 0.946

𝑅400𝐼Zn𝐶5 0.969 0.986 0.956

𝑅1000𝐼Fe𝐶20 0.925 0.985 0.950

𝑅400𝐼Fe𝐶20 0.955 0.986 0.958

(𝑃 > 0.05). As mentioned earlier Fe2+ ions produced biggerparticle size than Zn2+ ions (Figure 2). This aggregationprobably expelled the trapped drug out of the nanoparticlesand drug release was enhanced and consequently RE

24%was

increased (Figure 2).

3.5.Mucoadhesive Properties of Nanoparticles. Figure 1 showsthat the most effective factor on the mucoadhesive propertiesof nanoparticleswas the interaction of stirring rate and theiontype (RI) and at a less extent the interaction of the iontype and curing time (IC). As the process of mucoadhesionis a consequence of interactions between the mucin andmucoadhesive polymer of the nanoparticles, it is greatlydependent on the polymer structure including its charge.Figure 2 indicates that none of the studied parameters hadsignificant effect on the mucoadhesion of the nanoparticles(𝑃 > 0.05) and this property in all formulations was similar.In fact,mucoadhesion of all formulationswas 90.6-90.7% andtheir differences were negligible. This is due to the constantconcentration of the mucoadhesive polymer used in theproduction of nanoparticles.

There are many types of anionic polymers with mucoad-hesive properties like poly(acrylic acid) Carbopol 980,

Carbopol 974P, Carbopol 971P, polycarbophil, poly(metha-cryl acid) sodium salt, sodium alginate, sodium carboxy-methylcelullose, sodium hyaluronate (highly polymerizedgrade). Some years ago, Peppas and Buri [53] analyzed theexisting data and theories relevant to mucoadhesive poly-mers. They came to the conclusion that a number of polymercharacteristics are necessary for mucoadhesion which can besummarized as follows: (i) strong hydrogen-bonding groups(–OH, –COOH), (ii) strong anionic charges, (iii) highmolec-ular weight, (iv) sufficient chain ‘flexibility, (v) surface energyproperties favouring spreading onto mucus. In contrast, neg-ative charge and hydrogen-bonding capabilities are commonto presently knownmucoadhesives, but should not a priori begeneralized. Instead, positively charged polymeric hydrogelscould possibly develop additional molecular attraction forcesby electrostatic interactions with negatively charged mucosalsurfaces. In the present study as the concentration of themucoadhesive polymer used in production of nanoparticleswas constant the mucoadhesion of nanoparticles was notaffected by none of the studied variables (𝑃 > 0.05). However,this test was carried out to prove the mucoadhesive propertyof the nanoparticles.

3.6. Optimization of the Production Process of sCT LoadedNanoparticles. In many formulations, not just pharmaceu-tical in nature, it is necessary to balance several differentmeasures of quality in order to find the best overall product.Changes in the formulation to improve one property mayhave a deleterious impact on another property. The processof finding the best compromise has been more rigorous bythe process of desirability optimization, to produce numericalvalue of a desirability function.

Computer optimization process by Design Expert soft-ware and a desirability function determined the effect ofthe levels of independent variables on the responses. Theconstraint of particle size was 166.5 ≤ 𝑌

1≤ 553.8 nm while

targeting the particle size on minimum: for zeta potential itwas −22.3 ≤ 𝑌

2≤ −14.6mV while the target was set on

maximum of zeta potential absolute values, for drug loading


Table 5: Comparison of the Design Expert predicted and actual values of responses studied in sCT loaded P(MVEMA) nanoparticles.

Response Particle size(nm)

Zeta potential(mV)

Drug loadingefficiency

(%)

Releaseefficiency(RE24 %)

Mucoadhesiveefficiency

(%)Predicted 188.3 −21.0 92.7 45.1 90.67Actual 196.1 ± 6.9 −19.7 ± 1.7 92.2 ± 2.5 49.5 ± 2 90.66 ± 0.04

Error % 4.1 6.2 0.5 9.9 0.01

17.00 kV12.5mm

45.00KX

17.00 kV12.5mm

45.00KX

200nm300nm

200nm

200nm

Cursor width = 170.6nm

Figure 4: SEM micrographs of the optimized nanoparticles of sCT.

efficiency the constraint was 87 ≤ 𝑌3≤ 93% with the

goal set at the range of obtained data (Table 3), the drugrelease percent after 24 h, or RE

24% constraint was 42.2 ≤

𝑌4≤ 54.8% with the target set at the maximum values of

the obtained data of Table 3 and for mucoadhesive percentconstraint was 90.6 ≤ 𝑌

5≤ 90.7 with the target set at the

maximum. Considering the data in Table 3 optimization wascarried out by Design Expert software and the optimizedformulation was suggested as 𝑅

500𝐼Zn𝐶5,that is, using stirring

rate of 500 rpm, Zn ion as the cross-linking agent, and curingtime of 5min. The predicted and actual values of responsesand their standard errors are recorded in Table 5.

Table 5 shows the actual results were in close accordancewith the predicted values by the software.

3.7. Morphological Studies. Morphology of the optimumformulation was studied by SEM (Figure 4). The SEM pho-tographs show smooth and compact structure of nanopar-ticles. In general, the nanoparticles are obviously discretesome spherical and some irregularly shaped and the scalebar of the graphs confirms that the particle sizes measuredby the Malvern Nanosizer instrument (Table 3) were com-parable with SEM results. However, slightly aggregation ofnanoparticles may be due to high bioadhesive properties ofP(MVEMA) polymer.

3.8. SDS-PAGE Studies. The stability of sCT during produc-tion and release test after 24 h was studied by SDS-PAGEtest. The results of representative samples and unstainedmolecular weight protein markers are shown in Figure 5.The protein marker samples are distinctly separated andseen in 6 bands. Also, according to concentration of theimplanted samples on the gel, an obvious band and a bandwith lower resolution were detected for standard sample(with 250𝜇gmL−1 concentration) and experimental sampleof 24 h release medium of the optimized formulation with58.1 𝜇gmL−1 concentration.

3.9. Pharmacological Effect of sCT Nanoparticles. The in vivopharmacological action of the prepared sCT nanoparticleswas evaluated by pulmonary administration of the dispersionto male Wistar rats. As reference formulations, iv injec-tion of sCT solution (5 𝜇g kg−1), pulmonary sCT solution(25 𝜇g kg−1), and blank nanoparticles dispersion (withoutsCT) were also included in the study. The pulmonary dose ofsCT in the form of nanoparticles dispersion was 25 𝜇g kg−1.Considering that the rats weighted 185–330 g and their meanweight was 257.5 g, the mean dose of sCT administered viapulmonary route was 6.4𝜇g and via iv injection it was 1.3 𝜇g.The final formulation of nanoparticles of sCT administered


116.0

66.2

45.0

32.0

25.0

18.4

14.4

3.5

(kD

a)1 2 3 4

Figure 5: A 20% SDS-PAGE slab gel stained with Coomassieshowing (1) unstainedmolecular weight proteinmarkers: 116.0 kDa;𝛽-galactosidase (E. coli), 66.2 kDa; bovine serum albumin (bovineplasma), 45.0 kDa; ovalbumin (chicken egg white), 35.0 kDa; lac-tate dehydrogenase (porcine muscle), 25.0 kDa; REase Bsp981 (E.coli), 18.4 kDa; 𝛽-lactoglobulin (bovine milk), 14.4 kDa; lysozyme(chicken egg white); (2) 24 h release medium of the optimizedsCT nanoparticles (58.1 𝜇g mL−1); (3) blank sample of 24 h releasemedium of the optimized nanoparticles but without sCT; and (4)standard sample of sCT solution (250 𝜇gmL−1).

to rats consisted of 250mg of P(MVEMA), 125mg ZnSO4,

and 2.5mg sCT in 7.5mL deionized water. In other words, theratio of P(MVEMA) to sCT in the nanoparticles dispersionadministered to rats was 100 : 1. Consequently the mean massof polymer administered to each animal at the sCT dosetested was 640 𝜇g in pulmonary and 130 𝜇g in iv injectionroute.

The results of blood calcium levels after sCT adminis-tration in different groups are summarized in Figure 6. Thisfigure shows pulmonary administration of sCT nanoparti-cles exhibited superior hypocalcemic effect compared to itspulmonary solution and iv administration. After pulmonaryadministration of sCTnanoparticles, the blood calcium levelsdecreased to 59%of normal level and this lasted until 24 h anda significant difference in the blood calcium concentrationwas observed with other groups for 24 h. In contrast, thehypocalcemic effect in the rats receiving pulmonary sCTsolution persisted for 3 h, which is approximately similar tothat caused by iv injection of sCT solutionwhich lasted for 1 h.A possible explanation for the prolongation pharmacologicaleffect of P(MVEMA) nanoparticles is that the nanoparticlesdispersion was more viscous than the sCT solution evencompared at the same concentration. The prolonged andcontrolled release of sCT from nanoparticles along with themucoadhesion of this dispersionmay partly contribute to theprolongation of the residence time of the nanoparticles inthe pulmonary tract, leading to more effective absorption.In conclusion, the mucoadhesion and sCT release propertiesfrom the system are key factors in promoting the absorptionof sCT in the lungs. This result agrees well with previousreports [54–57]. We should also pay attention to stabilityof peptide drugs in the pulmonary tract. The peptide drugsare susceptible to enzymatic or chemical degradation. The

40

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80

90

100

110

120

0 4 8 12 16 20 24 28 32 36 40 44 48

Redu

ctio

n in

blo

od ca

lciu

m le

vel (

%)

Time (hr)

iv sCL solutionPulmonary sCL solution

Blank sCL nanoparticlesPulmonary sCL nanoparticles

Figure 6: Comparison in reduction of blood calcium levels afteradministration of sCT in different groups of rats (𝑛 = 7, sCT doseof iv solution = 5 𝜇g kg−1, pulmonary dose of sCT in the form ofnanoparticles or solution = 25 𝜇g kg−1).

P(MVEMA) nanoparticles can also protect sCT againstdegradation in the pulmonary tract. It has also been reportedthat some polymers such as poly(acrylic acid) derivatives andchitosan were able to inhibit the activities of the proteolyticenzymes [57, 58].

The minimum blood calcium levels, the duration ofhypocalcemia, and the area under the reduction of bloodcalcium levels after administration of sCT solution and sCTnanoparticles dispersion are summarized in Table 6. sCT wasrapidly eliminated from the plasma after its intravenous injec-tion in rats. The concentration of sCT in plasma decreasedgradually after pulmonary administration and after 24 hoursdecreased the blood calcium level to approximately 59 ± 5%,but after 48 hours it reached back to baseline. In contrast,the concentration of sCT in plasma decreased rapidly afterintravenous administration.

Finally, the overall pharmacological effect of pulmonaryadministered sCT nanoparticles was evaluated by calculatingthe areas above the curve (AAC) of reduced blood calciumlevels up to 48 h. The results are seen in Table 7. To considerthe dose of the sCT in the iv and pulmonary administration,the normalization was carried out by dividing the AAC tothe administered dose in different routes (Table 7). This tableindicates the pulmonary sCT nanoparticles caused 2.76 timeshigher effect (AAC/dose) than that of pulmonary adminis-tered free sCT solution. However, no significant difference(𝑃 > 0.05) was seen in pharmacological action (AAC/dose)between the pulmonary sCT solution (25 𝜇g kg−1) and iv(5 𝜇g kg−1) administered solution of this drug (Table 7).

The relative short duration of action of pulmonarysolution of sCT may be the result of rapid elimination ofdrug by mucociliary clearance and/or drug degradation bypulmonary peptidases [59]. On the other hand, prepared sCTnanoparticles significantly prolonged the hypocalcemic effect


Table 6: Duration of hypocalcemic effect and maximum reduction in blood calcium levels after pulmonary and iv administration of sCTsolution and sCT nanoparticles dispersion. Results are expressed as mean ± SE of seven rats.

Studied parameter Intravenous injectionof sCT solution

Pulmonary administration

sCT solution sCT nanoparticlesdispersion

Dose (𝜇g kg−1) 5 25 25Blood calcium level compared to normallevel (%) 72 ± 7.5 82.5 ± 9.5 59 ± 5

Duration of hypocalcemia (h) 0.5–1 3-4 12–24AAC0–48(% h) 211.0 ± 63.0 302.1 ± 100.9 1259.9 ± 147.2

Table 7: Pharmacodynamics of sCT after iv and pulmonary administration to rats.

Formulation Dose (𝜇g kg−1) AAC0–48 h (% h) AAC

0–48 h/dose(% h kg𝜇g−1)

Pharmacologicalactivity relative to iv

route (%)iv solution of sCT 5 211.05 ± 63.03 42.21 ± 12.61 —Pulmonary sCT solution 25 302.08 ± 100.93 12.08 ± 4.04

¥28.63 ± 9.56

Pulmonary blank nanoparticles 25 25.10 ± 5.56 1.00 ± 0.22 2.4 ± 0.53

Pulmonary sCT nanoparticles 25 1259.95 ± 147.19 50.40 ± 5.89∗¥

119.4 ± 13.95

∗Significant difference (𝑃 < 0.001) between iv solution of sCT and pulmonary sCT nanoparticles, ¥significant difference (𝑃 < 0.001) between pulmonary sCTsolution and pulmonary sCT nanoparticles.

of sCT with approximate pharmacological activity relative toiv route of 119% (Table 7).

Moreover, partial protection against enzymatic degrada-tion is expected due to the protecting effect of the nanoparti-cles.The higher efficacy and longer duration of hypocalcemiceffect of the prepared nanoparticles could be the result of thehigher mucoadhesive properties of the P(MVEMA) polymeras mentioned earlier.

Comparison of the results of the current study withprevious findings should be made with caution becauseof the differences in the in vivo model, dose, sCT type,administration method, and evaluation procedure. However,P(MVEMA) polymer and its derivative should be consideredas potentially safe and effective polymer for enhancing thepulmonary delivery of peptide drugs. Zinc ion (cross linkingreagent) is generally regarded as biologically compatible andsafe material as compared to the other ions. Additionally,formulation of sCT nanoparticles offers an appropriate sizefor avoiding alveolar macrophage clearance and promotingtransepithelial transport in the alveolar region.

4. Conclusion

The pulmonary sCT nanoparticles of P(MVEMA) wereprepared for increasing hypocalcemic action and enhancingthe duration of their hypocalcemic effect. The cross-linkingion used in their preparation caused reproducible results.The stirring rate, ion type, and curing time were detectedas effective variables in production of the sCT nanoparticles.The best formulation of the sCT nanoparticles was preparedby stirring rate of 500 rpm, ion type of Zn as cross-linkingagent, and the curing time of 5min.Wedemonstrated that the

pharmacological availability of pulmonary sCTnanoparticleswas approximately 3-fold higher than that of pulmonarysCT solution in rats. Furthermore, it was confirmed that theelimination rate of sCT loaded nanoparticles from the lungswas decreased significantly due after pulmonary adminis-tration compared to that of the free sCT. The sCT loadednanoparticles resulted in a pronounced hypocalcemic effectfor at least 24 h and a corresponding reduction in plasmacalcium level of approximately 59%. Inhaled sCT nanoparti-cles improved hypocalcemic control over that of intravenoussCT. However, further in vivo pharmacokinetic and toxicitystudies should be performed to check the effectiveness andsafety of the developed formulation.Moreover; future studieswill establish whether or not this suggestion will be verifiedin the marketplace.

Conflict of Interests

The authors declare that there is no conflict of interestregarding the publication of this paper.

Acknowledgments

The authors acknowledge the financial support of IsfahanUniversity of Medical Sciences. This paper is extracted fromthe dissertation of Mozhgan Forghanian Khan the PharmDstudent of Faculty of Pharmacy of Isfahan University ofMedical Sciences.

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